Figure 4.

P-S65-Ub sandwich ELISA from cultured cells treated with mitochondrial depolarizers. (A) WT HEK293 cells, PRKN KO and PINK1 KO HEK293 cells were treated with mitochondrial depolarizers (20 μM CCCP, 1 μM valinomycin, the combination of 10 μM oligomycin and 4 μM antimycin) or vehicle for 24 h. p-S65-Ub levels were determined by sandwich ELISA and representative western blots are shown below the graph. (B) PD patient-derived human skin fibroblasts carrying no mutation (WT), compound heterozygous PRKN mutations p.R275W/ΔExon2 or homozygous PINK1 p.Q456X mutations were treated with 1 μM valinomycin for 0, 4, 8, or 24 h. PINK1 KO HEK293 cells treated with AP were used as additional negative control. p-S65-Ub levels were determined by sandwich ELISA and representative western blots are shown below the graph. MSD-ECL values (mean + Std. Dev.) are shown from three replicates for both p-S65-Ub ELISAs in A and B. Two-Way ANOVA and Tukey’s post-hoc test (* p < 0.05, ** p = 0.005, *** p < 0.0001). Asterisks (*) indicate the comparison to WT samples for the same treatment (A) or time point (B). Number signs (#) indicate the comparison to the control treatment within the same genotype. Plus signs (+) indicate comparison between PRKN and PINK1 mutant genotypes